Expression of the vnd/NK-2 homeobox gene initiates the neural pathway of development in the ventral neuroectoderm that gives rise to part of the ventral nerve cord of Drosophila embryos. Previously we showed that -8.4 to +0.35 kb of DNA from the 5-flanking region of the vnd/NK-2 gene, ligated to a beta-galactosidase (B-gal) reporter gene in a P- element results in a pattern of B-gal in transgenic embryos similar to the wild-type pattern of vnd/NK-2. During the past year additional lines of transgenic Drosophila were generated. Embryos from transgenic lines of Drosophila with -5.3 to -2.2 kb of DNA from the 5-flanking region of the vnd/NK-2 gene inserted in the P-element, pCaSpeR-hs43- LacZ, expressed B-gal in a pattern similar to that of wild-type vnd/NK- 2 and ectopically in a cluster of unidentified cells. Similar results were found with transgenic flies containing -5.3 to -2.9 kb of DNA from the 5 flanking region of the vnd/NK-2 gene. However, transgenic lines of flies with either -5.3 to -3.5, -5.3 to -4.0, or -4.0 to -2.9 kb of DNA from the 5-flanking region of the vnd/NK-2 gene expressed B-gal in partial vnd/NK-2 patterns. Embryos with -4.0 to -2.9 kb of DNA from the 5 regulatory region of the vnd/NK-2 gene also expressed B-gal ectopically in a cluster of unidentified cells. Fragments of DNA from the 5-upstream region of the vnd/NK-2 gene were subcloned in a vector that contains an enhancerless chloramphenicol acetyltransferase (CAT) reporter gene. Transfection of Schneider 2 cells with constructs containing -0.75 to -0.41 kb from the 5 flanking region of the vnd/NK-2 gene activated expression of the CAT gene one hundred and ninety fold; whereas, co-expression with either constitutively expressed vnd/NK-2 or snail cDNA markedly repressed CAT gene expression. The three- dimensional structure of the vnd/NK-2 homeodomain-DNA complex was determined by NMR spectroscopy in collaboration with James Ferretti and his colleagues. Hydrogen bonding was found between Asn51 and the second adenine of the DNA binding site. Hydrogen bonding by Arg5 and Lys3 in the minor groove of DNA appears to be responsible for two unusually upfield-shifted ribose H1 resonances. Interactions were described involving Leu7, Thr41, and Gln50 of the vnd/NK-2 homeodomain, where single amino acid residue mutations in other homeodomains result in dramatic developmental alterations. Structural changes in the vnd/NK-2 homeodomain induced by binding to DNA were identified. - vnd/NK-2 homeobox gene, Drosophila, pattern formation, development, central nervous system, gene regulation, protein-DNA interaction